US9709501B2 - Cytological method using the auto fluorescence of white corpuscles for the early diagnosis and the monitoring of infections - Google Patents

Cytological method using the auto fluorescence of white corpuscles for the early diagnosis and the monitoring of infections Download PDF

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US9709501B2
US9709501B2 US14/006,455 US201214006455A US9709501B2 US 9709501 B2 US9709501 B2 US 9709501B2 US 201214006455 A US201214006455 A US 201214006455A US 9709501 B2 US9709501 B2 US 9709501B2
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Karim Asehnoune
Marie-Pierre Fontaine-Aupart
Sandrine Lecart
Antoine Monsel
Antoine Roquilly
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Centre National de la Recherche Scientifique CNRS
Universite de Nantes
Universite Paris Saclay
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Universite de Nantes
Universite Paris Sud Paris 11
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G01N15/1463—
    • G01N2015/008—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • Severe sepsis or serious bacterial infection remains a major cause of hospital morbidity-mortality, more specifically in intensive care units. Furthermore, its current incidence of 3 to 11 cases per 1,000 inhabitants in the United States has increased by 8.7% per year over the past ten years. 14.6% of admissions to French intensive care units are related to this pathology. In spite of many improvements in diagnostic and therapeutic procedures, hospital mortality related to severe sepsis is estimated at 35% in France. It is now clear that the time period separating the admission of the patient from the initiation of the antibiotic therapy constitutes a major prognostic factor of mortality. The goal is thus, in the 21 st century, to diagnose the infection as early as possible, which is a gauge of patient survival.
  • TLRs Toll-like receptors
  • TLRs have the common characteristic of leading to the activation of the nuclear transcription factor NF- ⁇ B and the production of NF- ⁇ B dependent proinflammatory cytokines, such as tumor necrosis factor alpha (TNF- ⁇ ) and interleukin-6 (IL-6).
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-6 interleukin-6
  • fMLP N-formyl-L-methionyl-L-leucyl-L-phenylalanine
  • This peptide a product of the breakdown of bacterial proteins, activates a G protein coupled receptor leading to a cascade of activation of intracytoplasmic kinases resulting in the phosphorylation of subunits whose assembly forms NADH-oxidase.
  • This membrane enzyme complex enables monocytes and polymorphonuclear neutrophils (PNN) to produce reactive oxygen species (ROS) by oxidizing NADH to NAD. These ROS participate in the bactericidal activity of phagocytes.
  • the present Inventors show herein that it is possible to use, in quite particular experimental conditions, the autofluorescence of these white blood cells to reveal the early immunological activity which is established during the microbial infection (and thus the infectious state of an individual).
  • the diagnostic method of the invention uses a routine optical material making it possible to work in wavelength ranges compatible with cellular autofluorescence, and thus constitutes a rapid, reliable and inexpensive aid to the diagnosis of an infection in an individual. It also makes it possible to very rapidly measure the effectiveness of anti-infection therapies established beforehand.
  • the present invention first relates to an in vitro method for diagnosing the infectious state of an individual based on a sample of white blood cells arising from a biological specimen from said individual, comprising at least the following steps:
  • the white blood cells of said sample are selected from monocytes and/or polymorphonuclear neutrophils.
  • said individual when the state of said individual is infectious, said individual is suffering from a bacterial, viral or fungal infection, preferably bacterial.
  • said biological specimen is a fluid arising from a potentially infected organ of said individual; more preferably this sample is a pulmonary fluid, ascites fluid, cerebrospinal fluid, blood sample or any other biological fluid arising from a potentially infected organ.
  • the autofluorescence intensity of the cells of said sample is measured on chemically fixed cells, by means of a fluorescence microscope, a flow cytometer, a spectrofluorometer or any other optical device capable of measuring fluorescence.
  • the sample of cells is prepared in a monolayer on slide of transparent material, preferably glass, prior to step i), according to a method comprising at least the following steps:
  • the state of said individual is infectious if the autofluorescence intensity measured in step i) is significantly greater than the control value.
  • the state of said individual is infectious if the autofluorescence intensity measured in step i) is significantly lower than the control value.
  • FIG. 1 consists of two images (A, B) taken by a confocal microscope, revealing the autofluorescence of monocytes and polymorphonuclear neutrophils arising from cytological slides of bronchoalveolar lavage (BAL) in a murine model of Staphylococcus aureus pneumonia.
  • FIG. 2 represents the variations of the autofluorescence intensity (I f ) as a function of time of human monocytes according to various stimulation conditions.
  • FIG. 3 illustrates the autofluorescence intensity (I f ) of monocytes-macrophages and PNN sampled in bronchoalveolar lavages (BAL, in black) in murine models of Pseudomonas aeruginosa or Staphylococcus aureus pneumonia. Background noise is represented by bars.
  • the method of the present invention uses the autofluorescence of special cells of the immune system in order to diagnose the presence of bacterial and/or viral agents in an individual at risk. Infectious bacterial and/or viral pathogens are capable, via the activation of TLRs, of causing early metabolic activity in these immune cells. This metabolic activity may be correlated with variations in the concentration of NAD(P)H, one of the essential coenzymes involved in cellular energy metabolism, characterized by its intracellular autofluorescence (Mayevsky A et al., Am J. Physiol. Cell Physiol. 2007).
  • the signal studied is a signal of undulatory variation of the autofluorescence intensity in time and space on a temporal scale of 0.1 ⁇ s to 20 ⁇ s, and on a spatial scale of a few microns.
  • the space-time undulatory changes thus take place in an infinitesimal temporal interval succeeding the in vitro immune stimulation of PNN (several ⁇ s after stimulation by the immune agonist concerned).
  • the second study relates to the demonstration of the effect of tobacco on the autofluorescence of macrophages of the lung. Pauly et al. have indeed identified that smoking increases the intensity of this autofluorescence (Pauly J. L. et al., Microsc Res Tech 2005).
  • the international patent application WO 99/50642 describes a method for diagnosing infections, based on the exploitation of the autofluorescence of whole plasma of patients suffering from a viral infection (AIDS virus or hepatitis virus) to establish spectral emission characteristics distinguishing the plasma of sick patients from that of controls.
  • the method of the present invention does not exploit the autofluorescence of plasma (as proposed by WO99/50642), but the autofluorescence of specific cells, namely immune cells (and, preferably, among these cells, monocytes and polymorphonuclear neutrophils) arising from whole blood or potentially infected organs.
  • the present Inventors have developed for the first time an experimental protocol making it possible to exploit the autofluorescence of white blood cells, and in particular the modulations of this autofluorescence related to their activation. They thus propose a reproducible, reliable and effective system for evaluating the infectious state of target individuals.
  • the present invention thus relates to an in vitro method for diagnosing the infectious state of an individual based on a sample of immune cells arising from a biological specimen from said individual, said method comprising at least the following steps:
  • the state of an individual when the state of an individual is “infectious,” said individual is infected with a microorganism, preferably a bacterium, virus or fungus (yeast or filamentous). In a more preferred manner, said individual suffers from a bacterial infection. This state can already have been diagnosed before and the individual can be subjected to an anti-infection treatment (antibiotic). In this case, the diagnostic method of the invention is intended to evaluate if the infection is still present and/or if the treatment can be stopped.
  • a microorganism preferably a bacterium, virus or fungus (yeast or filamentous).
  • “individual” refers to an animal or a mammal, and in particular man.
  • a “biological” specimen is defined in the present invention as any specimen of biological fluid, preferably arising from potentially infected organs and thus containing white blood cells. It is possible in the context of the present invention to take for example a sample of pulmonary fluid (in the case of pneumonia), of ascites (in infections of ascites fluid), of cerebrospinal fluid (in meningitis) or of blood, or of any other potentially infected organ.
  • obtaining a sample of cells arising from a biological specimen consists of 1) taking a sample of biological fluid from an individual, preferably from an organ of an individual, and 2) specifically in the context of a blood sample, purifying from this sample the cells of interest by conventional cell biology methods, in order to obtain the sample of purified cells.
  • the optional step of cellular purification to preferentially obtain cells of interest is not necessary when a biological fluid other than blood is used. Indeed, for the other sampled fluids (pulmonary, cerebrospinal or ascites fluid), in the case of infection, these fluids will contain in large majority cells of interest (PNN and monocytes), and thus there is no need to purify them.
  • the volume taken is preferably at least 10 ml to 20 ml, so as to obtain sufficient cells of interest after purification.
  • a sample of a fluid from an organ a few ml are enough (500 ⁇ l to 1 ml per cytospin well).
  • the sample of cells that can be used in the diagnostic method of the invention can be either preserved at room temperature to be used extemporaneously (in practice, less than 5 hours after the sample is taken) or preserved at a temperature of 4° C. in order to preserve the integrity of the cells until the operations of cell purification/isolation, fixing and/or measurement of autofluorescence intensity.
  • the “mean cellular autofluorescence intensity” measured in step i) of the diagnostic method of the invention is the total autofluorescence intensity for a cell suspension or a median cell value obtained on at least about 50 cells, preferably about 100 cells. It is then advisable to 1) subtract from this total value the value of autofluorescence due to possible contaminating cells, and 2) divide this remaining intensity by the number of cells of interest (monocytes or polymorphonuclear neutrophils) present in the sample studied. This final value will be the mean cell intensity in the context of the present invention.
  • control value (or “standard”) to which it is advised to compare the autofluorescence intensity of the individual to be tested is the mean cellular autofluorescence intensity obtained from a large number of isolated cells arising from several healthy individuals not having had treatment for at least 7 days (notably neither antibiotic nor anti-inflammatory treatment), not exhibiting any declared detectable infection (no signs or symptoms of infection, such as, for example: fever, aches, pains, etc.).
  • This control value is typically calculated on at least 100 cells isolated from at least 5 healthy individuals. It is calculated beforehand using the standard experimental parameters that will be used for the patient tested (notably in terms of optical material used, cell fixation method, excitation and emission wavelengths, temperature, pH, etc.).
  • Immune cells also called “white blood cells” or “leukocytes,” are human blood cells containing a mono or multi-lobed nucleus, which play essentially a role in the defense of the organism against foreign agents in the context of innate immunity.
  • white blood cells mononuclear cells (B-cells, T-cells, monocytes, macrophages) are distinguished from polymorphonuclear cells (or “granulocytes,” which include neutrophils, eosinophils and basophils) (Keneth M. Murphy, Paul Tavers, & Mark Walport, Janeway's Immunobiology. 7 th Edition)
  • the white blood cells of said sample are selected from monocytes and/or polymorphonuclear neutrophils.
  • the cell sample contains preferably at least about 80%, preferably about 90% of PNN, still more preferably at least about 95% of PNN.
  • Certain biological fluids taken directly from organs are known to contain only PNN.
  • fluids from bronchoalveolar lavage as well as from ascites contain mostly PNN (E. Pilly, Maladies infect (2004)s et tropicales, 22 nd edition, 2010). In this case a preliminary purification step will not be necessary.
  • the sample of biological fluid used in the present invention does not undergo a purification step because it naturally contains at least about 80%, preferably about 90% of PNN, still more preferably at least about 95% of PNN.
  • the sample of biological fluid is thus preferentially a sample of BAL, ascites or cerebrospinal fluid (CSF).
  • a purification step were required to obtain a cell sample that can be used in the method of the invention (notably in the case of a blood sample)
  • a sample of polynucleated cells from a biological fluid, it is advisable to use one of the techniques well-known to persons skilled in the art. For example, mention may be made of the density gradient technique and then withdrawal of the lower band, which corresponds essentially to polymorphonuclear cells (see the Cederlane®, Tebu-bio technique described in the examples below).
  • neutrophilic formulas mostly PNN. However, some are mostly monocytic or mixed (50/50). These proportions depend on the pathogenic agent responsible for the pneumonia, on the individual and/or on the more or less advanced stage of pneumonia. All the biological fluids referred to in the invention (blood, pulmonary fluid, cerebrospinal fluid, ascites fluid) can contain mostly monocytes in certain particular cases of infection.
  • the cell sample thus contains preferably at least about 80%, preferably about 90% of monocytes, still more preferably at least about 95% of monocytes.
  • the sample of biological fluid used in the present invention does not undergo a purification step because it naturally contains at least about 80%, preferably about 90% of monocytes, still more preferably at least about 95% of monocytes (in the case of pulmonary fluid, cerebrospinal fluid or ascites fluid).
  • monocytes can be isolated from mononucleated cells by techniques well-known to persons skilled in the art. For example, positive selection by separation on a magnetic column (anti-CD14 antibody) can be carried out.
  • positive selection by separation on a magnetic column anti-CD14 antibody
  • cell biology techniques well-known to persons skilled in the art. For example, the density gradient technique can be used, and then the upper band comprising these cells can be withdrawn (see the Cederlane®, Tebu-bio technique described in the examples below).
  • Fluorescence is the property in which a molecule emits a photon in order to return from its excited state (following the absorption of another photon) to its ground state.
  • Cells contain molecules that can become fluorescent when they are excited with rays of wavelengths in the visible or ultraviolet (UV) range.
  • This emission of fluorescence, emanating from endogenous fluorophores is an intrinsic property of cells called “autofluorescence” or “endogenous fluorescence” and must be distinguished from the fluorescence signals obtained by the addition of exogenous markers.
  • the known fluorophores are: aromatic amino acids, lipophilic dyes, NADPH, flavins and porphyrins. Autofluorescence does not require specific marking (Monici M., Biotechnol Annu Rev. 2005).
  • the light source used in the method can thus take any form of optical tool as long as it is capable of providing a sufficiently powerful intensity of light in the wavelength range concerned.
  • LASER UV (argon) sources are capable of providing an incidental beam with such characteristics
  • other optical tools capable of providing wavelength ranges compatible with the excitation of cellular NADH can also be used. This is the case with certain lamps (mercury, halogen, etc.) of LASER diode and white LASER lamps (delivering a continuous emission spectrum over a broad range of wavelengths).
  • cells in suspension i.e., cells that are not attached to a support and that move freely in a suitable liquid medium
  • cells fixed on a transparent support preferably a glass slide.
  • the autofluorescence intensity of the cells of said sample is measured on cells in suspension.
  • the autofluorescence can be measured with systems that measure fluorescence without an observer being able to see these cells, for example by flow cytometry, spectrophotometry or microspectrofluorimetry.
  • the mean autofluorescence intensity is measured automatically by systems (flow cytometers, spectrophotometers) that measure the total fluorescence of each sample.
  • the autofluorescence intensity of the cells of said sample is measured on chemically fixed cells placed on a slide compatible with the observation of fluorescence, preferably a transparent glass slide, and the autofluorescence is then measured with conventional fluorescence microscopy (epifluorescence microscopy), confocal microscopy or two-photon microscopy apparatuses.
  • fluorescence microscopy epifluorescence microscopy
  • confocal microscopy confocal microscopy or two-photon microscopy apparatuses.
  • the mean autofluorescence intensity is then measured by means of image processing software, the image being obtained from a suitable device such as a camera coupled to a microscope.
  • a flow cytometer a conventional spectrophotometer, or any type of microscope making it possible to obtain a fluorescence signal using intensity imaging or photon counting can be used.
  • the autofluorescence intensity is measured for cells fixed on a transparent glass slide, using a conventional epifluorescence microscope.
  • the autofluorescence intensity of the cells of said sample is measured using an epifluorescence microscope that excites the cells with a wavelength ranging from about 300 nm to about 600 nm, preferably ranging from about 350 nm to about 450 nm. This excitation is carried out preferably by using a laser UV source.
  • the autofluorescence intensity of the cells of said sample measured in step i) of the present invention is preferably that emitted at a wavelength ranging from about 400 nm to about 700 nm, preferably ranging from about 450 nm to about 600 nm.
  • the autofluorescence intensity of the cells of said sample is measured in step i) of the diagnostic method of the invention using a confocal fluorescence microscope with a continuous argon UV laser source, a photomultiplier sensitive in the wavelength range of about 400 nm to about 550 nm, an open pinhole and a X63/1.4 oil immersion objective.
  • the mean cellular autofluorescence intensity is measured on living cells.
  • these living cells were isolated from an organ, and were resuspended preferably in a liquid medium in a culture device that can be transferred to an optical exploitation tool (such as the cell culture well or LabTek® used in the first experimental phase described below).
  • an optical exploitation tool such as the cell culture well or LabTek® used in the first experimental phase described below.
  • the present Inventors have shown that it was important, to measure autofluorescence on living cells in a reliable and reproducible manner, to maintain the pH and the temperature of the culture medium at a constant value (pH preferably between 7 and 8, still more preferably 7.4, and temperature maintained at 37° C.) throughout the measurement of autofluorescence. Otherwise, the autofluorescence intensity values measured will be less reliable and less reproducible.
  • the cellular autofluorescence intensity of the sample is measured on cells chemically fixed, i.e., fixed in a particular cellular metabolic state, for example using paraformaldehyde.
  • the cells of the biological specimen are chemically fixed as quickly as possible, preferably less than 5 hours, after the biological sample is taken.
  • the white blood cells in a certain state, it is instantaneous cellular metabolic state that is studied. This makes it possible to avoid all the technical difficulties related to the perpetuation of a living cellular state which a method studying living cells would require. This is why it is important to minimize time between the fixing of the cells of the sample and the taking of the sample.
  • the present Inventors tested several fixing methods and several slide mounting systems in order to obtain a reliable measurement of autofluorescence for the two types of immune cells below (monocytes and PNN, see example 3 below). In this way, they identified a common cell sample treatment protocol suited to the constraints of the method of the invention, i.e., one that ensures a sufficient autofluorescence signal making it possible to obtain reliable and reproducible results for these two cell types.
  • the cells must be prepared in a monolayer by cytocentrifugation on a glass microscope slide before being fixed on said slide with paraformaldehyde (PFA), washed, dried, and then finally mounted in a mounting medium compatible with the observation of fluorescence before being covered with a glass cover slip.
  • PFA paraformaldehyde
  • the sample of cells is prepared in a monolayer on a slide of transparent material, preferably made of glass, prior to step i), according to a method comprising at least the following steps:
  • step i) of measuring the mean cellular autofluorescence intensity of the sample is performed using a fluorescence microscope.
  • the number of cells introduced into the well of the centrifugation system is advantageously between 500,000 and 1,000,000 in 500 ⁇ l to 1 ml of volume per well.
  • cytospin-type cytocentrifugation system for example, that marketed by Thermo Electron Corporation under the name Shandon Cytospin®
  • Shandon Cytospin® a cytospin-type cytocentrifugation system
  • Cytocentrifugation is today a well-known technology that makes it possible to deposit cells of interest placed in the well in a well-defined area on a microscope slide of transparent material, preferably made of glass, in a monolayer, and allows the absorption of residual liquid by the sample chamber filter.
  • the rotatory movement of the instrument tilts the wells in a straight position and centrifuges the cells onto the deposition area of the slide, providing all the cells with the same possibility of being exposed.
  • the transparent slide (or “microscope slide”) on which the cells of interest are deposited is preferably a glass slide suited to the analysis of cellular fluorescence, of thickness ranging between 1.2 mm and 1.5 mm, preferably 1.5 mm, such as those marketed by Fisher Scientific.
  • step a) The device obtained in step a) is then centrifuged. This centrifugation of step b) is carried out between 400 and 1000 rpm, preferably at about 600 rpm (41 g) for about 5 minutes.
  • step c the microscope slide is then taken out of the centrifuge, and the cell spot obtained after step b) is chemically fixed with paraformaldehyde (PFA) solution, diluted in PBS solution at a concentration ranging between about 2% and 6%, preferably about 4%.
  • PFA paraformaldehyde
  • the quantity of PFA is advantageously about 15 ⁇ l of paraformaldehyde (PFA) for about 10 6 cells, or a 1 ml drop of PFA on the cell spot projected on the slide.
  • PFA paraformaldehyde
  • the PFA fixing time is between 2 minutes and 20 minutes, and is preferably about 10 minutes.
  • the fixed cell spot is then rinsed, for example with PBS, to eliminate the remaining PFA (step d)). Three washes are generally required in order to effectively eliminate the remaining PFA.
  • step e the cell spot is dried completely, for example with air, for the required time, about 10 minutes at room temperature.
  • the cell spot is then covered with a drop (preferably 20 ⁇ l) of a mounting medium compatible with the observation of fluorescence.
  • This medium is, for example, the “ProLong® Gold” or “Fluoromount” mounting medium, or “Vectashield Slow Fade.”
  • this medium is “ProLong® Gold.”
  • cover slip suitable to the measurement of fluorescence, preferably made of glass, typically from 0.13 to 0.17 mm in thickness, such as those offered by Fisher Scientific.
  • the present Inventors have shown that, by comparing the mean cellular autofluorescence intensity of the tested patient's sample with the so-called “control” value, when the white blood cells of said sample are mostly monocytes, the state of said individual is infectious if the autofluorescence intensity measured in step i) is significantly greater than the control value.
  • a sample of cells comprising at least about 70%, preferably at least about 80%, still more preferably at least about 90% of monocytes emits an autofluorescence signal significantly greater than the control value, then the individual from whom the sample of biological fluid was taken is suffering from (or is still infected by) a bacterial, viral or fungal infection.
  • the term “significantly greater” means, in the context of the present invention, that the ratio [mean cellular intensity of the patient]/[control value] is at least about 1.2, advantageously between about 1.3 and 3, in a preferred manner between about 1.5 and 2.
  • the present Inventors have shown that, conversely, by comparing the mean cellular autofluorescence intensity of the sample from the patient tested with the so-called “control” value, when the white blood cells of said sample are mainly polymorphonuclear neutrophils, the state of said individual is infectious if the autofluorescence intensity measured in step i) is significantly lower than the control value.
  • the difference in result between the two cell populations can be explained by, among other things, the very different metabolic nature of these two cells.
  • a sample of cells comprises at least about 70%, preferably at least about 80%, still more preferably at least about 90% of polymorphonuclear neutrophils, and emits a autofluorescence signal that is significantly lower than the control value, then the individual from whom the sample of biological fluid was taken is suffering from (or is still infected by) a bacterial, viral or fungal infection.
  • the term “significantly lower” means, in the context of the present invention, that the ratio [mean cellular intensity of the patient]/[control value] is at most about 0.8, advantageously between about 0.1 and 0.7, in a preferred manner between about 0.1 and 0.6.
  • the present invention thus relates to a method for preparing a fixed cell sample intended to be used in the diagnostic method as described above, and comprising the following steps:
  • this method of preparation can be carried out automatically by means of robots capable of carrying out these steps sequentially.
  • This automated system has the advantage of being much faster than the microbiological techniques currently used and much less expensive than the genetic techniques aimed at identifying the presence of an infectious agent, which, moreover, remain not completely validated notably in the case of bacterial infection.
  • Lympholyte-Poly® comes from Cedarlane Tebu-Bio® (Le Perrey-en-Yvelines, France).
  • the MACS® kit for insolating monocytes and anti-CD14 antibodies come from Miltenyi Biotec® (Paris).
  • Purified lipopolysaccharide (LPS) of Escherichia coli 0111: B4 comes from Sigma® (Saint Louis, Mo., United States of America).
  • Pam3Csk4 (hereafter PAM) comes from InvivoGen® (San Diego, United States of America).
  • fMLP comes from Sigma-Aldrich® (St Quentin Fallavier, France).
  • Sterile observation chambers for Lab-Tek® confocal microscopy come from Brands Products® (United States of America). Sterile 24-well plates and conical tubes come from Falcon®, Becton Dickinson Labware (Europe, Le Pont de Claix, France). The specific mounting medium ProLong® Gold Antifade reagent comes from Invitrogen (ref.: P36934). The centrifuge used for cytocentrifugation and to project the cell samples on a slide is a Shandon Cytospin® centrifuge.
  • the microscope available at the imaging unit of the biomedical photonics center of Orsay is a Leica® TCS SP5® confocal microscope. It has of 4 continuous lasers (2 helium-neon lasers, 633 nm and 543 nm, a visible argon laser, 458 nm, 476 nm, 488 nm and 514 nm, and an argon UV laser, 351 nm and 364 nm) and a high pulse rate titanium/sapphire infrared laser.
  • the first acquisition mode used is that of the analysis of fluorescence intensity (I f ) quantified in AU.
  • a mean of 4 images with a resolution of 1024 pixels/1024 pixels for a final size of 246 ⁇ m/246 ⁇ m was applied to display the image in I f .
  • MNC mononucleated cells
  • a density gradient technique well-known to persons skilled in the art (Lympholyte-Poly®, Cedarlane Tebu-Bio®, Le Perrey-en-Yvelines, France). This technique makes it possible to obtain two distinct bands of cells, an upper band of MNC (lymphocytes and monocytes) which was withdrawn and a lower band of polynucleated cells, comprised essentially of polymorphonuclear neutrophils.
  • the monocytes were then isolated from the MNC using a positive selection technique with separation on a magnetic column (MACS®, Miltenyi Biotec, Paris).
  • the blood monocytes were then suspended in RPMI 1640+4% human serum+penicillin G (100 U/ml) and streptomycin (100 ng/ml) (Valbiotech).
  • the two cell populations thus obtained (PNN and monocytes) were incubated for 90 minutes in the presence of immune agonists with a cell density of 10 6 cells/ml.
  • LPS was used at a final concentration of 50 ⁇ g/mL
  • PAM at a final concentration of 10 ⁇ g/mL
  • fMLP at a final concentration of 10 ⁇ 6 mol/l.
  • the cytology slides thus obtained were then observed using a confocal laser scanning microscope.
  • a mean of 4 images at a resolution of 1024 pixels/1024 pixels for a final size of 246 ⁇ m/246 ⁇ m was applied to display the image in I f .
  • MSSA methicillin-sensitive Staphylococcus aureus
  • a wild-type Pseudomonas aeruginosa strain was grown for 18 hours at 37° C. in tryptic soy liquid medium. Immediately before the intratracheal instillation, the cultures were washed twice (centrifuged for 10 minutes at 5000 g at 37° C.) and diluted in sterile isotonic serum to be calibrated by spectroscopy, followed by calibration by nephelometry to obtain a concentration of 1 ⁇ 10 6 CFU/ml. The control is prepared on Cetrimide agar (selective medium for Pseudomonas aeruginosa ).
  • mice were anaesthetized with isoflurane, and were placed in the decubitus dorsal position.
  • An enteral feeding needle 24 gauge was used for the catheterization of the trachea and the injection of 70 ⁇ l of bacterial solution ( Staphylococcus or Pseudomonas aeruginosa ).
  • the mice were then suspended by the incisors for 30 seconds to improve the penetration of the inoculum.
  • the intratracheal instillation rate reached 100%.
  • a bronchoalveolar lavage was carried out by catheterizing the trachea by percutaneous route (24 gauge catheter). Washes with 3 ⁇ 1 ml of physiological saline solution were then carried out. A cell count using a Malassez counting chamber was then carried out. The alveolar lavage fluid was then centrifuged (10 minutes at 5000 g at 37° C.) and the cell pellet was resuspended in physiological saline solution q.s. 1 ⁇ 10 6 cells/ml.
  • a 500 ⁇ l volume of cell sample (5 ⁇ 10 5 cells) was delicately withdrawn and deposited in a well coupled to a microscopy slide in accordance with the Cytospin® device.
  • a centrifugation-slide projection cycle was carried out at 600 rpm for 8 minutes with a low acceleration-deceleration.
  • the cell spot projected on the slide was fixed with a drop of 4% paraformaldehyde (PFA) for 10 minutes followed by 3 delicate rinses with PBS buffer solution. After complete drying, a 20 ⁇ l drop of ProLong® Gold Antifade reagent mounting medium freshly unfrozen was deposited on the fixed cell spot.
  • a glass cover slip was then delicately placed on the drop of mounting medium while taking care to avoid any formation of air bubbles.
  • a highly significant increase in the number of PNN cells in relation to macrophages is observed in the mice having been inoculated with one or the other of the bacterial solutions ( Staphylococcus or Pseudomonas aeruginosa ).
  • the percentages of polymorphonuclear neutrophils observed in the infected animals in relation to the control animals (sham) confirm the presence of a bacterial pneumonopathy.
  • the sample slides thus obtained were then observed using a confocal laser scanning microscope.
  • a mean of 4 images at a resolution of 1024 pixels/1024 pixels for a final size of 246 ⁇ m/246 ⁇ m was applied to display the image in I f .
  • mice Twenty-nine mice were studied with 8 control mice, 9 mice with Pseudomonas aeruginosa pneumonia, and 12 mice with Staphylococcus pneumonia.
  • the mean cellular autofluorescence intensity decreases for the slides from the mice with pneumonia in relation to the cells from the BAL from the control mice.
  • the majority of cells (PNN) from the BAL of the mice with pneumonia fluoresce less than those from the BAL of the control mice (44.8 AU vs 107.5 AU).
  • a factor of 2 exists between the mean autofluorescence intensity of the cells from the BAL of the mice with pneumonia and the mean autofluorescence intensity of the cells from the BAL of the control mice.

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